The field of the invention is chemical modification of proteins and other biomolecules. Specifically, the present invention discloses engineered non-self-cleaving inteins of Mxe GyrA and methods of using such inteins to chemically modify proteins and other biomolecules.
Therapeutic and biochemical properties of proteins, such as antibodies, can be enhanced by custom chemical functionalization that enables modifications such as small molecule drug conjugation, PEGylation, and conjugation to nanoparticles. Expressed protein ligation (EPL) is one common approach to chemically modify proteins in a site-specific manner. In EPL, the target protein is expressed as a fusion partner to a non-self-cleaving intein such as Mxe GyrA.
Intein-mediated protein splicing is activated by the addition of a nucleophile, such as a thiol nucleophile, that releases the target protein from the intein while simultaneously producing a carboxy-terminal thioester intermediate on the target protein. Subsequently, this carboxy-terminal thioester can be reacted with an appropriately functionalized amino-terminal cysteine to covalently attach a desired moiety to the carboxy-terminus of the target protein.
Non-self-cleaving intein fusion proteins are most often expressed in the cytoplasm of Escherichia coli. One disadvantage of cytoplasmic expression is the formation of insoluble inclusion bodies that contain inactive protein-intein fusions, therefore requiring solubilization of the inclusion bodies and refolding of the protein. Glutathione redox buffers that are typically used to refold disulfide-containing proteins like antibodies can react with the thioester intermediate formed by the intein, thereby releasing it from the target protein and forming an unstable glutathione thioester on the carboxy-terminus of the target protein. This unstable glutathione thioester can subsequently be hydrolyzed leading to loss of the thioester functionality. Additionally, in vivo autocleavage of the intein has been observed during protein expression, resulting in up to 90% loss of the intein for some fusion proteins. These factors have combined to hamper protein-intein fusion protein production using bacteria.
Yeasts provide a possible alternative to bacterial expression systems, given their eukaryotic quality control machinery. In earlier work from Applicants' lab, scFvs were displayed as fusions to the Mxe GyrA intein on the surface of Saccharomyces cerevisiae. Contrasting with bacterial protein-intein fusion platforms, yeast-displayed scFv-intein protein fusions were properly folded and capable of engaging their antigenic targets. However, surface display levels of the scFvs were reduced by ˜40% when fused to intein compared to the unfused antibody. In addition, surface display of heterologous proteins is not ideally suited for protein production at a preparative scale as the yield is too low (˜70 μg of scFv/L).
Needed in the art are engineered non-self-cleaving inteins for significantly improving production of the resulting proteins with chemical functionalization. Specifically, needed in the art are engineered inteins and methods of using such inteins to improve yeast production of protein-intein fusions.